Difference between revisions of "Team:Linkoping Sweden/Experiments"

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<div class="column full_size">
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Overview on Laboration
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</div>
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<li> Week 1: 13 – 19 June </li>
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14 June: First day at the lab! Making Hutner’s trace elements 
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</div>
  
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<li> Week 2: 20 – 26 June  </li>
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21 June: Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates.
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46 agar plates were made.
  
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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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</div>
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<li> Week 3: 27 June – 3 July </li>
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27 June: Transformation of E1010 - The transformation was successful
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28 June: Control of competent cells
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29 June: Transformation of E1010 to super competent XL-1 - The transformation was successful.
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30 June: Making E.Coli Calcium Chloride competent cells
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1 July:
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-          Making solutions for TAP- and TRIS medium
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-          Cultivation of XL1 and E1010
  
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</div>
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<li> Week 4: 4 – 10 July </li>
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4 July:
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-          Making LB-medium and LB-agar.
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-          Plasmid preparation of E1010
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- Test cultivation of algae
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5 July:
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-          Making agar plates
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-          Digestion and ligation of LIP, U6, UTR and LIP-RFP.
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-          Transformation of E1010 and MD-cells competent test
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-    First algae cultivation
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6 July: Transformation on U6, LIP, LIP-RFP and UTR.
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Colonies for U6 and LIP were detected.
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7 July: Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol
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8 July: OD measurement of transformated bacteria.
 
</div>
 
</div>
  
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<li> Week 5: 11 – 17 July </li>
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<h5>What should this page contain?</h5>
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11 July: PCR on Cas9
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<ul>
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12 July: Gel electrophoresis on Cas9 to see if the PCR succeeded.
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<li> Protocols </li>
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The gel did not show any bands for Cas9
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<li> Experiments </li>
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13 July: PCR
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<li>Documentation of the development of your project </li>
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14 July: PCR on pSB1C3
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</ul>
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15 July:
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-          Gel electrophoresis on pSB1C3
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No bands were obtained on the gel.
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-          PCR on pSB1C3
  
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</div>
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<li> Week 6: 18 – 24 July </li>
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18 July: PCR and gel electrophoresis on pSB1C3
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No bands were obtained.
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20 July: PCR and gel electrophoresis on pSB1C3
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We obtained bands on the gel at approximately 2000 bp.
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22 July: Digestion and ligation on LIP, U6, UTR, Cas9, LIP-RFP, sgRNA and pSB1C3.
 
</div>
 
</div>
  
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<div class="column half_size">
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<li> Week 7: 25 – 31 July </li>
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<h5>Inspiration</h5>
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25 July:
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<ul>
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-          PCR on LIP and UTR from colonies
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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-          Cultivation of LIP and UTR colonies on new plates
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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-          PCR purification
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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26 July:
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</ul>
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-          Gel electrophoresis on pSB1C3, UTR and LIP
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No bands were obtained.
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-          Digestion and Ligation on LIP-RFP and pSB1C3.
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27 July: New project approach
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Transformation of LIP-RFP and pSB1C3.
 
</div>
 
</div>
  
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<li>Week 8: 1 – 7 August </li>
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1 August:
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-          Cultivation of Hyg
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-          Gel electrophoresis on UTR and LIP
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Bands were obtained at 700 bp.
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3 August:
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-          Plasmid preparation of LIP, UTR and Hyg.
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Was later show to be wrong
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-          Transformation of LIP-RFP, U6, sgRNA and Cas9.
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4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6.
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4 August: Making TAP medium for cultivation of algae in the dark
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</div>
  
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<div class="clear"></div>
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<li>Week 9: 8 – 14 August </li>
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8 August:
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-          PCR on LIP-RFP and sgRNA
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-          Cultivation of LIP-RFP and sgRNA colonies on new plates
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-    First algae cultivation in darkness
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9 August:
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-          Transformation of U6 and Cas9.
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-          Gel electrophoresis on LIP-RFP and sgRNA.
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No bands on the gel.
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10 August:
 +
-          PCR on LIP-RFP and sgRNA
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-          Gel electrophoresis on LIP-RFP
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Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good. 
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11 August:
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-          PCR on U6 and Cas9.
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-          Gel electrophoresis on sgRNA.
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Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp.
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12 August: Gel electrophoresis on Cas9 and U6.
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Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp.
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</div>
  
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<li>Week 10: 15 – 21 August </li>
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15 August:
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-          Preparation of TAP agar
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Because of difficulties with the gas no plates could be performed today.
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-          Gel electrophoresis on UTR, LIP, Hyg and pSB1C3.
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pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands.
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-          Cultivation of Hyg.
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16 August:
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-          Cultivation of sgRNA, LIP-RFP and U6.
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-          PCR on Cas9 and Hyg
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-          Gel electrophoresis on Cas9 and Hyg
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The gel showed a weak band on Cas9 around 4000 bp.
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17 August: Plasmid preparation on LIP-RFP, U6 and sgRNA
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Was later show to be wrong
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18 August:
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-          Cultivation of algae for transformation
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It took 5 days for the algae wild type to reach OD 1,757
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The mutant alga evaporated
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-          Making TAP agar plates
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-          Making TAP Hygromycin plates
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</div>
  
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<div class="column half_size">
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<li>Week 11: 22 – 28 August </li>
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22 August: Cultivation of LIP-RFP and Hyg
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23 August:
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-          Plasmid preparation on LIP-RFP and Hyg
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-          PCR on Cas9 and pSB1C3
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- New cultivation of algae in the dark
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24 August:
 +
-          PCR on LIP-RFP, Hyg and pSB1C3
 +
-          Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3.
 +
Bands for Cas9 and Hyg were obtained.
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-          PCR purification of Cas9.
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25 August:
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-          Gel electrophoresis on pSB1C3
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Bands were detected at 2000 bp which match with pSB1C3
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-          PCR purification on pSB1C3
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-          First Gibson Assembly!
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-          Transformation of Gibson Assembly
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Colonies were obtained!
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-          PCR on Cas9, Hyg, pSB1C3
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26 August:
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-          PCR on Gibson Assembly product and colonies from Gibson Assembly transformation
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-          Chloramphenicol plates
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52 plates were made!
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-          Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3
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Bands were detected for all the DNAs!
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</div>
  
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<li>Week 12: 29 August – 4 September </li>
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29 August:
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-          Gel electrophoresis on Gibson Assembly colonies
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A band at 5500 bp was obtained. We want bands at 7000 bp.
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-          Digestion on LIP-RFP
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-          PCR on Gibson Assembly colonies.
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30 August:
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-          PCR on Gibson Assembly colonies.
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-          Cultivation of Gibson Assembly coloni on new plates
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-          Ligation on LIP-RFP with pSB1C3.
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-          New Gibson Assembly transformation
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31 August:
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-          Gel electrophoresis on Gibson Assembly colonies
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No bands.
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-          Plasmid preparation on Gibson Assembly colony.
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1 September:
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-          PCR on plasmid prepared U6, UTR, LIP, LIP-RFP, sgRNA and Hyg.
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-          Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg.
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No result on the gel.
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2 September
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-          Second Gibson assembly
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-          Gibson transformation
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- Transformation LIP-RFP
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3 September
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- PCR  and gel electrophoresis on gibson colonies
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No results
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- Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3
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4 September
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- PCR and gel electrophoresis on gibson colonies
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It looks like Colony 8 has a band at 7000 bp! Yeeey!
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</div>
  
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<li>Week 13: 5 – 11 September </li>
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5 September
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PCR on old colonies of LIP-RFP
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Making LB-medium
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Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies.
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6 September
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Cultivation of colony 8 (Gibson Assembly) with Hyg in the LB-media
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Screening of colonies from Gibson Assembly
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Bands were obtained, but no band was at 7000 bp.
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7 September
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PCR and gel electrophoresis on Hyg
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8 September
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Plasmid preparation of Gibson Assembly colony 8
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Screening on Gibson colonies
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9 September
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The sequences were obtained
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We did not insert Hyg but instead YFP was inserted.
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Cas9 and LIP are inserted successfully!
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10 September
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Cultivation of algae mutants and Gibson colony 8
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Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies
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The plasmid preparation of Gibson colony 8 showed good bands.
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11 September
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PCR on some Gibson colonies
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Preparation for plasmid preparation
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The cells were centrifuged and the pellet was saved for later continuation of plasmid preparation
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Cultivation of Gibson Assembly colonies on new plates
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</div>
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<li>Week 14: 12 – 18 September </li>
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13 September
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OD measurments on the algae
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Gel electrophoresis on the PCR product from 11/9 - 16.
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Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp were obtained.
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14 September
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Dilution of the algae
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Making TAP-40mM sucrose
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Plasmid preparation of Gibson Assembly colony 8
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15 September
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Digestion of Gibson colony 8
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16 September
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Electroporation on algae
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The algae have grown well.
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17 September
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PCR on Gibson colonies
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Continuation on the electroporation from previous day.
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18 September
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Gel electrophoresis on Gibson colonies
  
 
</div>
 
</div>
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<li>Week 15: 19 – 25 September </li>
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19 September
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Sequenced was obtained
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Looks like we did not insert U6 and sgRNA :(
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21 September
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PCR on Gibson 3
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Gel electrophoresis on the PCR product from today
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Band were obtained at 300 bp and 2000 bp.
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22 September
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Gel electrophoresis on PCR product from yesterday
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Bands at 2000 bp and 300 bp were obtained
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Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively.
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Transformation on all the Gibson product
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23 September
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Gel electrophoresis on Gibson 3 colonies
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No bands.
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PCR of Gibson with LIP, LIP-RFP, U6 and Term.
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Screening of YFP transformed algae
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No proof that the transformation worked.
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24 September
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Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP
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Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No result for U6.
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Gel electrophoresis on Gibson 3 colonies
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No bands.
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PCR on Gibson with U6, Term, LIP and LIP-RFP
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Cultivation of U6, Term, LIP and LIP-RFP
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25 September
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PCR on Gibson 3 colonies
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Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP.
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Bands were obatined.
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Cultivation of U6, Term, LIP and LIP-RFP
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</div>
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 +
<li>Week 16: 26 – 30 September </li>
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26 September
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PCR on U6 colonies
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Gel electrophoresis on Gibson 3 colonies
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No result.
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Plasmid preparation on LIP, LIP-RFP and Term.
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27 September
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Plasmid preparation nr 2 on LIP, LIP-RFP and Term.
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Gel electrophoresis on U6 colonies
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No result.
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PCR on Gibson 3 colonies
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28 September
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Gel electrophoresis on Gibson 3 colonies
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No result.
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Cultivation of U6, Term, LIP and LIP-RFP
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PCR on Gibson 3 colonies.
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 +
 +
3 October
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Sekvensing of LIP, LIP-RFP and Term
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 +
  
 
</html>
 
</html>
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Revision as of 19:02, 5 October 2016

LiU iGEM
Team
Collaborations Attributions
Project
Design Experiments Protocols Results Safety Notebook Economic Viability
Project Description
CRISPR/Cas9 System Gibson Assembly Chlamydomonas Reinhardtii
Parts
Human Practices
Silver Gold
Overview on Laboration
  • Week 1: 13 – 19 June
    • 14 June: First day at the lab! Making Hutner’s trace elements
  • Week 2: 20 – 26 June
    • 21 June: Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. 46 agar plates were made.
  • Week 3: 27 June – 3 July
    • 27 June: Transformation of E1010 - The transformation was successful 28 June: Control of competent cells 29 June: Transformation of E1010 to super competent XL-1 - The transformation was successful. 30 June: Making E.Coli Calcium Chloride competent cells 1 July: - Making solutions for TAP- and TRIS medium - Cultivation of XL1 and E1010
  • Week 4: 4 – 10 July
    • 4 July: - Making LB-medium and LB-agar. - Plasmid preparation of E1010 - Test cultivation of algae 5 July: - Making agar plates - Digestion and ligation of LIP, U6, UTR and LIP-RFP. - Transformation of E1010 and MD-cells competent test - First algae cultivation 6 July: Transformation on U6, LIP, LIP-RFP and UTR. Colonies for U6 and LIP were detected. 7 July: Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol 8 July: OD measurement of transformated bacteria.
  • Week 5: 11 – 17 July
    • 11 July: PCR on Cas9 12 July: Gel electrophoresis on Cas9 to see if the PCR succeeded. The gel did not show any bands for Cas9 13 July: PCR 14 July: PCR on pSB1C3 15 July: - Gel electrophoresis on pSB1C3 No bands were obtained on the gel. - PCR on pSB1C3